Project Summary/Abstract Mutations play a fundamental role in the etiology of many diseases including cancers, diabetes, epilepsy, schizophrenia, and others. Many of these mutations result from translesion synthesis, a pathway the cell uses to bypass DNA damage during DNA replication. In translesion synthesis, the high-fidelity classical DNA polymerase (i.e., the one involved in normal DNA replication) encounters DNA damage and stalls. Low-fidelity non-classical polymerases are recruited to the stalled replication fork where they carry out replication of the damaged DNA. Each non-classical polymerase is specialized for incorporating nucleotides opposite a few types of DNA damage. When the proper non-classical polymerase is used, damage bypass is not mutagenic. When an improper one is used, it is mutagenic. The overall goal of the proposed research is to understand the factors that affect the accuracy and efficiency of translesion synthesis. We are particularly interested in examining how non-classical polymerases are selected and regulated during translesion synthesis. Little is known about non-classical polymerase selection and regulation, because these polymerases function within dynamic, multi-protein complexes (bypass complexes) that are difficult to study. We are using an innovative and novel combination of biochemical, biophysical, and structural approaches that will allow us to overcome these challenges. In Aim 1, we will examine the architecture of bypass complexes using single-molecule total internal reflection fluorescence (TIRF) experiments. In Aim 2, we will examine the regulation of bypass complexes using steady state and pre-steady state kinetics studies. In Aim 3, we will examine the structure of bypass complexes using X-ray crystallography, small-angle X-ray scattering (SAXS) and Brownian dynamics (BD) simulations.